Iron and sulfur reducing and/or oxidizing bacteria are important for use in the leaching of certain ores to extract heavy metals. The ability of these organisms to leach ores is due to the possession of unique enzyme systems that can oxidize and/or reduce both iron (Fe+2) and sulfur compounds. For example, acidophilic thiobacilli are major bacteria in the leaching of sulfide ores at low pH. These bacteria are able to derive energy and reducing power for growth from the oxidation of ferrous iron and inorganic sulfur compounds, such as elemental sulfur, sulfide, thiosulfate, and polythionate. Studies have also demonstrated that these organisms possess enzymes to reduce metals and inorganic sulfur compounds as well. Generally, these organisms can be acidophilic, autotrophic, and chemolithotrophic.
For example, Thiobacillus ferroxidans has two kinds of sulfur reducing enzyme systems, namely a pH 1.5 sulfur-reducing system and a pH 7.5 sulfur-reducing system which produce H2S from elemental sulfur. It has also been demonstrated that H2S is produced from tetrathionate via a two-step reaction by Thiobacillus ferrooxidans. This two step reaction comprises: 1) decomposition of tetrathionate by a tetrathionate-decomposing enzyme to provide elemental sulfur and trithionate, and 2) reduction of the elemental sulfur produced to H2S.
Zeolites are microsporous, aluminosilicate minerals used as commercial adsorbents. The term zeolite was originally coined in 1756 by Swedish mineralogist who observed that upon rapidly heating the material stilbite, it produced large amounts of steam from water that had been adsorbed by the material. Zeolites have a porous structure that can accommodate a wide variety of cations, such as Na+, K+, Ca2+, Mg2+ and others. These positive ions are rather loosely held and can readily be exchanged for others in a contact solution.
Recently, some imported dry wall from Chinese wall board manufacturers has been found to emit numerous noxious and toxic gases, such as hydrogen sulfide and approximately 10 other noxious gases, once the wall board has been exposed to atmospheric conditions of increased humidity and increased dew point. The wall board (e.g., dry wall) becomes moist and emits gases which cause respiratory problems, sinusitis, skin irritation, and vision problems. For example, these emitted gases can cause a disproportionate number of illnesses for the inhabitants of homes with the wall board. An explanation why the wall board (e.g., dry wall) exudes such gases is needed to be able to prevent these illnesses from occurring and to be able to treat patients with these illnesses.
Applicant has discovered iron and sulfur reducing and/or oxidizing bacteria in wall board, and these organisms are likely the cause of the gases exuded from the wall board with the resulting illnesses for the inhabitants of homes with the wall board. The bacterial contaminants that can inundate the indoor environment from these bacteria include substances such as microbial volatile organic compounds (MVOC), allergenic proteins, inorganic compounds and gases, and organic compounds and gases.
Bacteria have typically been identified based on growth characteristics on specific selective media, and by either biochemical or carbohydrate utilization of specific compounds. These identification methods are based on the knowledge that a specific genus or species of bacteria produce or do not produce enzymes that utilize specific chemicals or carbohydrates for growth. Because these methods may require specific media and growth on these media for days to weeks for an identification to be made, these methods are time consuming. These methods are also inaccurate because not all of the organisms in a sample will be culturable, inevitably leading to the possible misidentification of the organism.
Thus, the invention further provides methods and compositions for the specific, sensitive, and rapid detection and identification of iron and sulfur reducing and/or oxidizing bacteria in wall board, such as dry wall. Applicant has developed bacterial DNA extraction procedures from wall board, such as dry wall, and has supplemented those methods by developing detection and identification methods. The detection and identification methods employ amplification of DNA probes and primers that specifically and selectively amplify bacterial DNA isolated from samples of wall board, such as dry wall. Kits and compositions for detecting and identifying iron and sulfur reducing and/or oxidizing bacteria in wall board, such as dry wall, are also provided. For example, acidophilic thiobacilli can be detected. Furthermore, iron and sulfur reducing and oxidizing bacteria such as Acidothiobacillus (thiobacillus) ferrooxidans, Thiobacillus thiooxidans, Leptospirillium ferroxidans, Thiobacillus caldus, Sulfobacillus thermosulfidooxidans, and Desulfotomaculum ruminis can be detected.
The invention also provides methods and compositions for the specific, sensitive, and rapid detection and identification of iron and sulfur reducing and/or oxidizing bacteria in patient tissues and body fluids. The detection and identification methods employ amplification of DNA probes and primers that specifically and selectively amplify bacterial DNA isolated from patient tissues and body fluids. Kits and compositions for detecting and identifying iron and sulfur reducing and/or oxidizing bacteria in patient tissues and body fluids are also provided. For example, acidophilic thiobacilli can be detected and identified. Furthermore, iron and sulfur reducing and/or oxidizing bacteria such as Acidothiobacillus (thiobacillus) ferrooxidans, Thiobacillus thiooxidans, Leptospirillium ferroxidans, Thiobacillus caldus, Sulfobacillus thermosulfidooxidans, and Desulfotomaculum ruminis can be detected and identified.
The invention also provides a method of treating wall board with a zeolite to eliminate a sulfur and iron reducing and/or oxidizing bacterial species in the wall board. Illustratively, the zeolite can comprise elemental ions (e.g., Ca+2, Mg+2, etc.) that bind to the sulfur and iron reducing and/or oxidizing bacterial species and limit the growth of these organisms.
In one embodiment, multiple iron and sulfur reducing and/or oxidizing bacterial species can be identified in either wall board or patient tissues or body fluids using a PCR-based reaction, saving money and time while insuring that the methodology is highly specific and accurate.
In one illustrative embodiment, a method is provided of identifying a specific iron and sulfur reducing and/or oxidizing bacterial species in wall board. The method comprises the steps of extracting and recovering DNA of the bacterial species from the wall board, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the bacterial species, and specifically identifying the bacterial species.
In still another embodiment, a method is provided of determining if a patient is at risk for or has developed a disease state related to an infection with a specific iron and sulfur reducing and/or oxidizing bacterial species. The method comprises the steps of extracting and recovering DNA of the specific bacterial species from a tissue or body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the bacterial species, and specifically identifying the bacterial species.
In another illustrative embodiment, a method is provided of identifying a specific iron and sulfur reducing and/or oxidizing bacterial species in a patient tissue or a body fluid. The method comprises the steps of extracting and recovering DNA of the specific bacterial species from a tissue or body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the bacterial species, and specifically identifying the bacterial species.
In yet another embodiment, a kit is provided. The kit comprises components for the extraction and recovery of the DNA of a sulfur and iron reducing and/or oxidizing bacterial species from wall board. The kit can further comprise instructions for the extraction and recovery of the sulfur and iron reducing and/or oxidizing bacterial species from the wall board.
In still another illustrative embodiment, a kit is provided. The kit comprises components for identification of a sulfur and iron reducing and/or oxidizing bacterial species. The kit components for identification of the sulfur and iron reducing and/or oxidizing bacterial species can be selected from the group consisting of a purified nucleic acid comprising a sequence of SEQ ID NO: 1 to SEQ ID NO: 18, a heat stable DNA polymerase, a buffer, MgCl2, H2O, and instructions for use.
In another embodiment, a kit is provided. The kit comprises components for the extraction and recovery of a sulfur and iron reducing and/or oxidizing bacterial species from wall board and components for identification of the sulfur and iron reducing and/or oxidizing bacterial species. The kit can further comprise any of the components described in the preceding paragraphs.
In another embodiment, a purified nucleic acid is provided. The purified nucleic acid comprises a sequence of SEQ ID NO: 1 to SEQ ID NO: 18 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 18.
In still another embodiment, a purified nucleic acid is provided. The purified nucleic acid comprises a complement of a sequence of SEQ ID NO: 1 to SEQ ID NO: 18 or a sequence that hybridizes under highly stringent conditions to a complement of a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 18.
In yet another illustrative embodiment, a method of treating wall board to eliminate a sulfur and iron reducing and/or oxidizing bacterial species in the wall board is provided. The method comprises the steps of treating the wall board with a zeolite, and eliminating the sulfur and iron reducing and/or oxidizing bacterial species in the wall board.
The following embodiments are also contemplated:
1. A method of identifying a specific iron and sulfur reducing and/or oxidizing bacterial species in wall board, the method comprising the steps of:                extracting and recovering DNA of the bacterial species from the wall board,        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the bacterial species; and        specifically identifying the bacterial species.        
2. The method of clause 1 wherein the amplifying step is performed with a primer that hybridizes to the DNA.
3. The method of any one of clauses 1 or 2 wherein the wall board is dry wall.
4. The method of any one of clauses 1 to 3 wherein the DNA is amplified using PCR.
5. The method of clause 4 wherein the PCR is real-time PCR.
6. The method of any one of clauses 1 to 5 wherein the probe is fluorescently labeled.
7. The method of any one of clauses 2 to 6 wherein the primer is fluorescently labeled.
8. The method of any one of clauses 1 to 7 wherein the bacterial species is selected from the group consisting of Acidothiobacillus ferrooxidans, Thiobacillus thiooxidans, Leptospirillium ferroxidans, Thiobacillus caldus, and Desulfotomaculum ruminis. 
9. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 1, the forward primer comprises the sequence of SEQ ID NO: 2, and the reverse primer comprises the sequence of SEQ ID NO: 3.
10. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 4, the forward primer comprises the sequence of SEQ ID NO: 5, and the reverse primer comprises the sequence of SEQ ID NO: 6.
11. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 7, the forward primer comprises the sequence of SEQ ID NO: 8, and the reverse primer comprises the sequence of SEQ ID NO: 9.
12. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 10, the forward primer comprises the sequence of SEQ ID NO: 11, and the reverse primer comprises the sequence of SEQ ID NO: 12.
13. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 13, the forward primer comprises the sequence of SEQ ID NO: 14, and the reverse primer comprises the sequence of SEQ ID NO: 15.
14. The method of any one of clauses 1 to 8 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 16, the forward primer comprises the sequence of SEQ ID NO: 17, and the reverse primer comprises the sequence of SEQ ID NO: 18.
15. A method of identifying a specific iron and sulfur reducing and/or oxidizing bacterial species in a patient tissue or a body fluid, the method comprising the steps of:                extracting and recovering DNA of the bacterial species from the patient tissue or body fluid;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the bacterial species; and        specifically identifying the bacterial species.        
16. The method of clause 15 wherein the amplifying step is performed with a primer that hybridizes to the DNA.
17. The method of any one of clauses 15 or 16 wherein the patient tissue and the body fluid are selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
18. The method of any one of clauses 15 to 17 wherein the DNA is amplified using PCR.
19. The method of clause 18 wherein the PCR is real-time PCR.
20. The method of any one of clauses 15 to 19 wherein the probe is fluorescently labeled.
21. The method of any one of clauses 16 to 20 wherein the primer is fluorescently labeled.
22. The method of any one of clauses 15 to 21 wherein the bacterial species is selected from the group consisting of Acidothiobacillus ferrooxidans, Thiobacillus thiooxidans, Leptospirillium ferroxidans, Thiobacillus caldus, and Desulfotomaculum ruminis. 
23. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 1, the forward primer comprises the sequence of SEQ ID NO: 2, and the reverse primer comprises the sequence of SEQ ID NO: 3.
24. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 4, the forward primer comprises the sequence of SEQ ID NO: 5, and the reverse primer comprises the sequence of SEQ ID NO: 6.
25. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 7, the forward primer comprises the sequence of SEQ ID NO: 8, and the reverse primer comprises the sequence of SEQ ID NO: 9.
26. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 10, the forward primer comprises the sequence of SEQ ID NO: 11, and the reverse primer comprises the sequence of SEQ ID NO: 12.
27. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 13, the forward primer comprises the sequence of SEQ ID NO: 14, and the reverse primer comprises the sequence of SEQ ID NO: 15.
28. The method of any one of clauses 15 to 22 wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 16, the forward primer comprises the sequence of SEQ ID NO: 17, and the reverse primer comprises the sequence of SEQ ID NO: 18.
29. A kit comprising a purified nucleic acid with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 or with a complement of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18.
30. A kit comprising components for the extraction and recovery of a sulfur and iron reducing and/or oxidizing bacterial species from wall board.
31. The kit of clause 30 further comprising components for identification of the sulfur and iron reducing and/or oxidizing bacterial species.
32. The kit of clause 31 wherein the components for identification of the sulfur and iron reducing and/or oxidizing bacterial species are selected from the group consisting of a purified nucleic acid comprising a sequence of SEQ ID NO: 1 to SEQ ID NO: 18, a heat stable DNA polymerase, a buffer, MgCl2, H2O, and instructions for use.
33. The kit of clause 30 further comprising instructions for the extraction and recovery of the sulfur and iron reducing and/or oxidizing bacterial species from the wall board.
34. A purified nucleic acid comprising a sequence of SEQ ID NO: 1 to SEQ ID NO: 18 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 18.
35. A purified nucleic acid comprising a complement of a sequence of SEQ ID NO: 1 to SEQ ID NO: 18 or a sequence that hybridizes under highly stringent conditions to a complement of a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 18.